1. Field of the Invention
The present invention relates to a method of producing polyhydroxyalkanoate (may be abbreviated as “PHA” hereinafter) as a polyester by using a substituted alkane derivative as a raw material. More specifically, the present invention relates to a method of producing PHA by using microorganisms having the ability to produce PHA and accumulate it in cells by using a substituted alkane derivative as a raw material.
2. Description of the Related Art
It has been reported that many microorganisms produce poly-3-hydroxybutyric acid (may be abbreviated as “PHB” hereinafter) or other PHAs, and accumulate these polymers in cells (“Biodegradable Plastic Handbook”, edited by Society of Biodegradable Plastic Research, N•T•S Co., Ltd., pp. 178–197 (1995)). Like conventional plastics, these polymers can be used for producing various kinds of products by melt processing, and the like. Furthermore, the polymers are biodegradable, and are thus advantageously decomposed completely by microorganisms in nature. Therefore, unlike synthetic polymers, these polymers doe not pollute the environment. The above polymers also have excellent biocompatibility. Thus, it may be expected that they form soft medical materials and like.
It is known that the microorganism producible PHAs have various compositions and structures depending on the types of the microorganisms used for production, culture medium compositions, culture conditions, etc. Therefore, studies have been conducted to control the compositions and structures of PHAs mainly to improve their physical propertie.
For example, it is reported that a strain of Alcaligenes eutropus H16 (ATCC No. 17699) or its mutants produce copolymers of 3-hydroxybutyric acid (“3HB”) and 3-hydroxyvaleric acid (“3HV”) in various composition ratios by using various carbon sources for the culture (Japanese Patent Publication Nos. 6-15604, 7-14352 and 8-19227).
Japanese Patent Publication No. 2642937 discloses that a non-cyclic aliphatic hydrocarbon is added as a carbon source to a strain of Pseudomonas oleovorans ATCC 29347 to produce PHA having 3-hydroxyalkanoate having 6 to 12 carbon atoms as a monomer unit.
Japanese Patent Laid-Open No. 5-7492 discloses a method in which microorganisms of Methylobacterium sp., Paracoccus sp., Alcaligenes sp., and Pseudomonas sp. are contacted with a primary alcohol having 3 to 7 carbon atoms to produce a copolymer of 3HB and 3HV.
Japanese Patent Laid-Open Nos. 5-93049 and 7-265065 disclose that a strain of Aeromonas caviae is cultured with oleic acid and olive oil as carbon sources to produce a binary copolymer of 3HB and 3-hydroxyhexanoic acid (“3HHx”).
Japanese Patent Laid-Open No. 9-191893 discloses that a strain of Comamonas acidovorans IFO 13852 is cultured with gluconic acid and 1,4-butanediol as carbon sources to produce a polyester having 3HB and 4-hydroxybutyric acid as monomer units.
The above-described PHAs have alkyl groups in side chains, i.e., “usual PHA”. However, in considering a wide application of the microorganism producible PHAs, PHAs having substituents other than alky groups, for example, a phenyl group, and the like, which are introduced in side chains, are expected to be useful polyesters. Examples of other substituents include an unsaturated hydrocarbon, an ester group, an allyl group, a cyano group, a halogenated hydrocarbon, an epoxide, and the like. Particularly, PHA having an aromatic ring is extensively studied.
(a) PHA having a phenyl group or partially-substituted phenyl group
In Macromolecules, 24, 5256–5260 (1991), it is reported that Pseudomonas oleovorans produces PHA having 3-hydroxy-5-phenylvaleric acid as a unit by using 5-phenylvaleric acid as a substrate.
More specifically, it is reported that Pseudomonas oleovorans produces PHA containing 3HV, 3-hydroxyheptanoic acid, 3-hydroxynonanoic acid, 3-hydroxyundecanoic acid, and 3-hydroxy-5-phenylvaleric acid (abbreviated as “3HPV” hereinafter) as monomer units in a ratio of 0.6:16.0:41.1:1.7:40.6 in an amount of 160 mg per liter of culture medium (dry weigh ratio of 31.6% relative to cells) by using 5-phenylvaleric acid (abbreviated as “PVA” hereinafter) and nonanoic acid as substrates (molar ratio, 2:1; total concentration, 10 mmol/L). It is also reported that Pseudomonas oleovorans produces PHA containing 3HHx, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, and 3HPV as monomer units in a ratio of 7.3:64.5:3.9:24.3 in an amount of 200 mg per liter of culture medium (dry weigh ratio of 39.2% relative to cells) by using PVA and octanoic acid as substrates (molar ratio, 1:1; total concentration, 10 mmol/L).
Besides the above reports, related descriptions are also found in Makromol. Chem., 191, 1957–1965 (1990) and Chirality, 3, 492–494 (1991) in which a change in polymer physical properties due to the presence of a 3HPV unit is recognized.
In Macromolecules, 29, 1762–1766 (1996), it is reported that Pseudomonas oleovorans produces PHA having 3-hydroxy-5-(4′-tolyl) valeric acid as a unit by using 5-(4′-tolyl) valeric acid as a substrate.
In Macromolecules, 32, 2889–2895 (1999), it is reported that Pseudomonas oleovorans produces PHA having 3-hydroxy-5-(2′,4′-dinitrophenyl) valeric acid and 3-hydroxy-5-(4′-nitrophenyl) valeric acid as units by using 5-(2′,4′-dinitrophenyl) valeric acid as a substrate.
(b) PHA containing a phenoxy group or a partially-substituted phenoxy group
In Macromol. Chem. Phys., 195, 1665–1672 (1994), it is reported that Pseudomonas oleovorans produces a PHA copolymer of 3-hydroxy-5-phenoxy valeric acid and 3-hydroxy-9-phenoxynonanoic acid by using 11-phenoxyundecanoic acid as a substrate.
In Macromolecules, 29, 3432–3435 (1996), it is reported that Pseudomonas oleovorans produces PHA containing 3-hydroxy-4-phenoxy-n-burylic aid and 3-hydroxy-6-phenoxy-n-hexanoic acid as units from 6-phenoxyhexanoic acid, PHA containing 3-hydroxy-4-phenoxy-n-butyric acid, 3-hydroxy-6-phenoxy-n-hexanoic acid and 3-hydroxy-8-phenoxy-n-octanoic acid as units from 8-phenxyoctanoic acid, and PHA containing 3-hydroxy-5-phenoxy-n-valeric acid and 3-hydroxy-7-phenoxy-n-heptanoic acid as units from 11-phenoxyundecanoic acid. In this report, the yields of the polymers are extracted and are shown in Table 1.
TABLE 1Carbon sourceDry cell weightDry polymerYield(alkanoate)(mg/L)weight (mg/L)(%) 6-phenoxyhexanoic acid95010010.5 8-phenoxyoctanoic acid820901111-phenoxyundecanoic acid1501510
Japanese Patent Publication No. 2989175 discloses an invention relating to a homopolymer composed of a 3-hydroxy-5-(monofluorophenoxy)pentanoate (3H5(MFP)P) unit or a 3-hydroxy-5-(difluorophenoxy)pentanoate (3H5(DFP)P) unit, a copolymer comprising at least the 3H5(MFP)P unit or 3H5(DFP)P unit, and a method of producing these polymers by using Pseudomonas putida, Pseudomonas sp. for synthesizing the polymers.
These polymers are produced by the following two-stage culture method.                Culture time: first stage, 24 hours; second stage, 96 hours        The substrate used in each stage and the resultant polymer are shown below.            (1) Resultant polymer: 3-hydroxy-5-(monofluorophenoxy) pentanoate homopolymer            Substrates in the first stage: citric acid, yeast extract        Substrate in the second stage: monofluorophenoxy undecanoic acid            (2) Resultant polymer: 3-hydroxy-5-(difluorophenoxy) pentanoate homopolymer            Substrates in the first stage: citric acid, yeast extract        Substrate in the second stage: difluorophenoxy undecanoic acid            (3) Resultant polymer: 3-hydroxy-5-(monofluorophenoxy) pentanoate copolymer            Substrates in the first stage: octanoic acid or nonanoic acid, yeast extract        Substrate in the second stage: monofluorophenoxy undecanoic acid            (4) Resultant polymer: 3-hydroxy-5-(difluorophenoxy) pentanoate copolymer            Substrates in the first stage: octanoic acid or nonanoic acid, yeast extract        Substrate in the second stage: difluorophenoxy undecanoic acid        For the effect, a polymer having phenoxy groups with side chain terminals substituted by 1 to 2 fluorine atoms can be synthesized by assimilation of a medium-chain fatty acid having substituents, and stereoregularity and water repellency can be imparted to the polymer while maintaining a high melting point and good processability.        
Besides the fluorine-substituted polymer, cyano- or nitro-substituted polymers have also be studied.
Can. J. Microbiol., 41, 32–43 (1995) and Polymer International, 39, 205–213 (1996) disclose that PHA containing 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit is produced by using a strain of Pseudomonas oleovorans ATCC 29347 and a strain of Pseudomonas putida KT 2442, and octanoic acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as substrates.
Unlike general PHA having alkyl groups in side chains, the PHA disclosed in this report has aromatic groups in side chains, and thus is advantageous for obtaining a polymer having physical properties derived from the aromatic rings.
(c) PHA having a monomer unit containing a cyclohexyl group is expected to exhibit polymer physical properties different from those of PHA having a monomer unit containing a usual aliphatic hydroxyalkanoic acid. Examples of production with Pseudomonas oleovorans are reported in Macromolecules, 30, 1611–1615 (1997).
In this report, a strain of Pseudomonas oleovorans is cultured in a culture medium containing nonanoic acid and cyclohexyl butyric acid or cyclohexyl valeric acid to obtain PHA having a unit containing a cyclohexyl group and a unit derived from nonanoic acid (the ratio is unknown).
With respect to yield, it is reported that the ratio of nonanoic acid to cyclohexyl butyric acid was changed under the condition of a total substrate concentration of 20 mmol/L to obtain the results shown in Table 2.
TABLE 2Nonanoicacid:cyclohexylbutyric acidCDWPDWYieldUnit5:5756.089.111.8nonanoic acid,cyclohexylbutyricacid1:9132.819.314.5nonanoic acid,cyclohexylbutyricacid                CDW: Dry cell weight (mg/L), PDW: Dry polymer weight (mg/L), Yield: PDW/CDW (%)        
However, in this example, the polymer yield per liter of culture medium is insufficient, and the resultant PHA itself contains aliphatic hydroxyalkanoic acid derived from nonanoic acid in the monomer unit.
A new category has also been studied, in which PHA having appropriate functional groups in side chains is produced not only for simply changing the physical properties but also for creating a new function by using the functional group.
For example, in Macromolecules, 31, 1480–1486 (1996) and Journal of Polymer Science: Part A; Polymer Chemistry, 36, 2381–2387 (1998), it is reported that PHA having a unit having a vinyl group at a side chain terminal is synthesized, and then epoxidized with an oxidizing agent to synthesize PHA having highly reactive epoxy groups at side chain terminals.
A synthetic example of PHA having a unit containing a sulfide group other than a vinyl group, which is expected to produce high reactivity, is PHA reported in Macromolecules, 32, 8315–8318 (1999) in which a strain of Pseudomonas putida 27N01 produces a PHA copolymer of 3-hydroxy-5-(phenylsulfanyl) valeric acid and 3-hydroxy-7-(phenylsulfanyl) heptanoic acid by using 11-phenylsulfanyl valeric acid as a substrate.
As described above, microorganism producible PHAs having different compositions and structures can be obtained by changing the types of the microorganisms used for production, the medium compositions, and culture conditions. However, the above-described PHAs are produced only for improving physical properties as plastic.
On the other hand, the above-described “unusual PHA” having substituents introduced in side chains can be expected as a “functional polymer” having useful functions and properties due to the properties of the introduced substituents. Therefore, it is thought to be very useful and important to develop an excellent polymer having the above-described function and biodegradability, microorganisms capable of producing the polymer and accumulating the polymer in cells, and a biosynthetic method of effectively producing the polymer with a high purity.
A general method of producing “unusual PHA” having any of various groups introduced in side chains, i.e., PHA having a monomer unit represented by formula (7), with microorganisms comprises chemically synthesizing a substituted fatty acid represented by formula (8), which has a substituent to be introduced, supplying the fatty acid to microorganisms for culture, and then extracting the produced PHA, as disclosed in the above-descried report examples of Pseudomonas oleovorans.
wherein R represents at least one residue selected from the group consisting of residues each having an aromatic ring, and r represents any integer of 1 to 8.R—(CH2)s-CH—CH2—COOH  (8)wherein R represents at least one residue selected from the group consisting of residues each having an aromatic ring, and s represents any integer of 1 to 8.
However, in the above-described general PHA producing method comprising chemically synthesizing the substituted fatty acid as a substrate, and supplying the substituted fatty acid to the microorganisms, a carboxyl group of the substituted fatty acid is an active group in a chemical reaction, and thus chemical synthesis of the fatty acid is greatly restricted according to the type, number and position of the substituents introduced. Therefore, a complicated operation of protecting an active carboxyl group in a reaction step of chemical synthesis, and deprotecting the carboxyl group is frequently required, thereby necessitating a chemical reaction comprising several steps. Therefore, synthesis in an industrial production level is difficult, or synthesis requires much time, labor and cost.
However, if “unusual PHA” can be produced by using as a raw material a substituted alkane that can easily be chemically synthesized, as compared with the substituted fatty acid, the above-described problem can be possibly resolved.
Conventional examples of PHA production from alkane derivatives include examples of biosynthesis of PHA with microorganisms using, as starting materials, straight-chain alkanes and alkenes (alkanes containing double bonds) (Appl. Environ. Microbiol., 54, 2924–2932 (1988)), chlorinated alkanes (Macromolecules, 23, 3705–3707 (1990)), fluorinated alkanes (Biotechnol. Lett., 16, 501–506 (1994)), and alkanes containing acetoxy residues (Macromolecules, 33, 8571–8575 (2000)). There is no report of synthetic examples of PHA using an alkane having a residue containing an aromatic ring as a substituent.